Cellular location and activity of Escherichia coli RecG proteins shed light on the function of its structurally unresolved C-terminus

RecG is a DNA translocase encoded by most species of bacteria. The Escherichia coli protein targets branched DNA substrates and drives the unwinding and rewinding of DNA strands. Its ability to remodel replication forks and to genetically interact with PriA protein have led to the idea that it plays an important role in securing faithful genome duplication. Here we report that RecG co-localises with sites of DNA replication and identify conserved arginine and tryptophan residues near its C-terminus that are needed for this localisation. We establish that the extreme C-terminus, which is not resolved in the crystal structure, is vital for DNA unwinding but not for DNA binding. Substituting an alanine for a highly conserved tyrosine near the very end results in a substantial reduction in the ability to unwind replication fork and Holliday junction structures but has no effect on substrate affinity. Deleting or substituting the terminal alanine causes an even greater reduction in unwinding activity, which is somewhat surprising as this residue is not uniformly present in closely related RecG proteins. More significantly, the extreme C-terminal mutations have little effect on localisation. Mutations that do prevent localisation result in only a slight reduction in the capacity for DNA repair. © 2014 The Author(s)

A New LC-MS/MS Method for the Quantification of Endogenous and Vinyl Chloride-Induced 7-(2-Oxoethyl)Guanine in Sprague–Dawley Rats

Vinyl chloride (VC) is an industrial chemical that is known to be carcinogenic to animals and humans. VC primarily induces hepatic angiosarcomas following high exposures (≥50 ppm). VC is also found in Superfund sites at ppb concentrations as a result of microbial metabolism of trichloroethylene and perchloroethylene. Here, we report a new sensitive LC-MS/MS method to analyze the major DNA adduct formed by VC, 7-(2-oxoethylguanine) (7-OEG). We used this method to analyze tissue DNA from both adult and weanling rats exposed to 1100 ppm [13C2]-VC for 5 days. After neutral thermal hydrolysis, 7-OEG was derivatized with O-t-butyl hydroxylamine to an oxime adduct, followed by LC-MS/MS analysis. The limit of detection was 1 fmol and the limit of quantitation was 1.5 fmol on the column. The use of stable isotope VC allowed us to demonstrate for the first time that endogenous 7-OEG was present in tissue DNA. We hypothesized that endogenous 7-OEG was formed from lipid peroxidation and demonstrated the formation of [13C2]-7-OEG from the reaction of calf thymus DNA with [13C18]-ethyl linoleate (EtLa) under peroxidizing conditions. The concentrations of endogenous 7-OEG in liver, lung, kidney, spleen, testis and brain DNA from adult and weanling rats typically ranged from 1.0-10.0 adducts per 106 guanine. The exogenous 7-OEG in liver DNA from adult rats exposed to 1100 ppm [13C2]-VC for 5 days was 104.0 ± 23.0 adducts per 106 guanine (n=4), while concentrations in other tissues ranged from 1.0-39.0 adducts per 106 guanine (n=4). Although endogenous concentrations of 7-OEG in tissues in weanling rats were similar to those of adult rats, exogenous [13C2]-7-OEG concentrations were higher in weanlings, averaging 300 adducts per 106 guanine in liver. Studies on the persistence of [13C2]-7-OEG in adult rats sacrificed 2, 4, and 8 wks post exposure to [13C2]-VC demonstrated a half-life of 7-OEG of 4 days in both liver and lung

Effect of remote ischaemic conditioning on clinical outcomes in patients with acute myocardial infarction (CONDI-2/ERIC-PPCI): a single-blind randomised controlled trial.

BACKGROUND: Remote ischaemic conditioning with transient ischaemia and reperfusion applied to the arm has been shown to reduce myocardial infarct size in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). We investigated whether remote ischaemic conditioning could reduce the incidence of cardiac death and hospitalisation for heart failure at 12 months. METHODS: We did an international investigator-initiated, prospective, single-blind, randomised controlled trial (CONDI-2/ERIC-PPCI) at 33 centres across the UK, Denmark, Spain, and Serbia. Patients (age >18 years) with suspected STEMI and who were eligible for PPCI were randomly allocated (1:1, stratified by centre with a permuted block method) to receive standard treatment (including a sham simulated remote ischaemic conditioning intervention at UK sites only) or remote ischaemic conditioning treatment (intermittent ischaemia and reperfusion applied to the arm through four cycles of 5-min inflation and 5-min deflation of an automated cuff device) before PPCI. Investigators responsible for data collection and outcome assessment were masked to treatment allocation. The primary combined endpoint was cardiac death or hospitalisation for heart failure at 12 months in the intention-to-treat population. This trial is registered with ClinicalTrials.gov (NCT02342522) and is completed. FINDINGS: Between Nov 6, 2013, and March 31, 2018, 5401 patients were randomly allocated to either the control group (n=2701) or the remote ischaemic conditioning group (n=2700). After exclusion of patients upon hospital arrival or loss to follow-up, 2569 patients in the control group and 2546 in the intervention group were included in the intention-to-treat analysis. At 12 months post-PPCI, the Kaplan-Meier-estimated frequencies of cardiac death or hospitalisation for heart failure (the primary endpoint) were 220 (8·6%) patients in the control group and 239 (9·4%) in the remote ischaemic conditioning group (hazard ratio 1·10 [95% CI 0·91-1·32], p=0·32 for intervention versus control). No important unexpected adverse events or side effects of remote ischaemic conditioning were observed. INTERPRETATION: Remote ischaemic conditioning does not improve clinical outcomes (cardiac death or hospitalisation for heart failure) at 12 months in patients with STEMI undergoing PPCI. FUNDING: British Heart Foundation, University College London Hospitals/University College London Biomedical Research Centre, Danish Innovation Foundation, Novo Nordisk Foundation, TrygFonden

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Avoiding chromosome pathology when replication forks collide

Chromosome duplication normally initiates through the assembly of replication fork complexes at defined origins. DNA synthesis by any one fork is thought to cease when it meets another travelling in the opposite direction, at which stage the replication machinery may simply dissociate before the nascent strands are finally ligated. But what actually happens is not clear. Here we present evidence consistent with the idea that every fork collision has the potential to threaten genomic integrity. In Escherichia coli this threat is kept at bay by RecG DNA translocase and by single-strand DNA exonucleases. Without RecG, replication initiates where forks meet through a replisome assembly mechanism normally associated with fork repair, replication restart and recombination, establishing new forks with the potential to sustain cell growth and division without an active origin. This potential is realized when roadblocks to fork progression are reduced or eliminated. It relies on the chromosome being circular, reinforcing the idea that replication initiation is triggered repeatedly by fork collision. The results reported raise the question of whether replication fork collisions have pathogenic potential for organisms that exploit several origins to replicate each chromosome

MatP regulates the coordinated action of topoisomerase IV and MukBEF in chromosome segregation

The Escherichia coli SMC complex, MukBEF, forms clusters of molecules that interact with the decatenase topisomerase IV and which are normally associated with the chromosome replication origin region (ori). Here we demonstrate an additional ATP-hydrolysis-dependent association of MukBEF with the replication termination region (ter). Consistent with this, MukBEF interacts with MatP, which binds matS sites in ter. MatP displaces wild-type MukBEF complexes from ter, thereby facilitating their association with ori, and limiting the availability of topoisomerase IV (TopoIV) at ter. Displacement of MukBEF is impaired when MukB ATP hydrolysis is compromised and when MatP is absent, leading to a stable association of ter and MukBEF. Impairing the TopoIV-MukBEF interaction delays sister ter segregation in cells lacking MatP. We propose that the interplay between MukBEF and MatP directs chromosome organization in relation to MukBEF clusters and associated topisomerase IV, thereby ensuring timely chromosome unlinking and segregation